US5374560A - Method for screening and distinguishing between cobalamin and folic acid deficiency based on assay for cystathionine and 2-methylcitric acid - Google Patents

Method for screening and distinguishing between cobalamin and folic acid deficiency based on assay for cystathionine and 2-methylcitric acid Download PDF

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US5374560A
US5374560A US07/727,628 US72762891A US5374560A US 5374560 A US5374560 A US 5374560A US 72762891 A US72762891 A US 72762891A US 5374560 A US5374560 A US 5374560A
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methylcitric
body fluid
methylcitric acid
cystathionine
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Robert H. Allen
Sally P. Stabler
John Lindenbaum
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/525—Isoalloxazines, e.g. riboflavins, vitamin B2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7135—Compounds containing heavy metals
    • A61K31/714—Cobalamins, e.g. cyanocobalamin, i.e. vitamin B12
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/13—Tracers or tags
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/18—Sulfur containing
    • Y10T436/182—Organic or sulfhydryl containing [e.g., mercaptan, hydrogen, sulfide, etc.]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/20—Oxygen containing
    • Y10T436/200833—Carbonyl, ether, aldehyde or ketone containing
    • Y10T436/201666—Carboxylic acid
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Definitions

  • This invention relates to a method of diagnosing cobalamin deficiency and folic acid deficiency and distinguishing between the two, in warm-blooded animals, particularly humans, by measuring serum levels of cystathionine and 2-methylcitric acid.
  • cobalamin and folic acid deficiency are often difficult because both deficiencies lead to indistinguishable hematologic abnormalities; the distinction is important because use of the proper vitamin results in the greatest improvement in hematologic abnormalities and, more importantly, only cobalamin will correct the neuropsychiatric abnormalities which are only seen in cobalamin deficiencies.
  • the use of folic acid to treat cobalamin deficiency is extremely dangerous, since some or all of the hematologic abnormalities may improve, but neuropsychiatric abnormalities will not improve and may progress, or even be precipitated.
  • hematological or neurological symptoms that were necessary to justify an assay for serum cobalamin and the resulting risk of a false positive, were fairly severe. Those symptoms included significant anemia, displayed for example in decreased hematocrit or hemoglobin, with macrocytic red blood cells (i.e., mean cell volume (MCV) generally greater than 100 fl), or neurologic symptoms of peripheral neuropathy and/or ataxia.
  • MCV mean cell volume
  • Anemia associated with cobalamin deficiency was described as typically severe with hemoglobin ⁇ 8 g % or hematocrit ⁇ 25% and the size of the red blood cells was described as greatly increased to levels >110 fl.
  • the two assays are relatively inexpensive and accurate, and can be performed concurrently by taking a single serum sample.
  • the assay described in the Allen patent is a very important test procedure but may not be infallible in detecting either kind of deficiency or in distinguishing between the two.
  • About 5-15% of the cobalamin deficiencies will not be detected because these patients will not show elevated homocysteine levels.
  • About 5-10% of the folic acid deficiencies will not be detected because these patients will not show elevated homocysteine levels.
  • elevated homocysteine Levels are in fact detected, thereby indicating either a cobalamin deficiency or folic acid deficiency, about 5-10% of the cobalamin deficiencies can not be distinguished from the folic acid deficiencies because the patients will not show elevated methylmalonic acid levels. Therefore the assays may produce a small but not insignificant number of false negative results.
  • the present invention uses an assay for cystathionine as a test for the existence of either of the two deficiencies or as a check on an assay for total homocysteine when testing for the two deficiencies. It also uses an assay for 2-methylcitric acid as a test for distinguishing between the two deficiencies or as a check on an assay for methylmalonic acid in distinguishing between the two deficiencies.
  • cystathionine is present in the urine and serum of children and adults with inherited defects of cystathionase who cannot convert cystathionine to cysteine and alpha-ketobutyrate.
  • the serum levels of cystathionine range from undetectable to as high as 80,000 nanomoles per liter.
  • Serum levels of methionine, homocysteine and cysteine are usually not elevated in these patients. See, e.g., Mudd, S. H., H. L. Levy and F. Skovby, in The Metabolic Basis of Inherited Disease, 6th Ed. (C. R. Scriver, A. L. Beaudet, W. S. Sly and D.
  • Elevated levels of cystathionine are present in the urine and serum of some children with inherited defects involving methionine synthase, who cannot convert homocysteine and N 5 -methyltetrahydrofolate to methionine and tetrahydrofolate, respectively. These patients usually have low levels of methionine, high levels of total homocysteine, and normal levels of cysteine in their serum.
  • cystathionine which tended to cast doubt on the reliability of such an indicator. See, e.g., Goodman, S. I., P. G. Moe, K. B. Hammond, S. H. Mudd, and B. W. Uhlendorf, Biochem. Med 4:500-515, 1970; Barshop, B. A., J. Wolff, W. L. Nyhan, A. Yu, C. Pordanos, G. Jones, L. Sweetman, J. Leslie, J. Holm, R. Green, D. W. Jacobsen, B. A. Cooper, and D. Rosenblatt, Am. J. Med. Genet.
  • the present invention measures cystathionine levels in the serum or urine and measures 2-methylcitric acid levels in the serum, urine or cerebral spinal fluid for the purpose of predicting cobalamin and folic acid deficiencies and for distinguishing between the two deficiencies. It has been determined that elevated cystathionine levels tend to correspond with the existence of either such deficiency, while elevated 2-methylcitric acid levels tend to correspond with the existence of cobalamin deficiency but not folic acid deficiency and can thereby be used to distinguish between the two deficiencies.
  • samples of serum or urine in the assay for cystathionine, and samples of serum, urine or cerebral spinal fluid in the assay for 2-methylcitric acid are mixed by vortexing with water and known amounts of stable isotope forms of cystathionine or 2-methylcitric acid, respectively.
  • the mixtures are washed, incubated, eluted and dried in the manner described herein, and then are analyzed by gas chromatography/mass spectrometry.
  • the assays for cystathionine and 2-methylcitric acid may be combined with assays for homocysteine or methylmalonic acid or both. Further, while the assays are described for use with serum or urine in the case of cystathionine and with serum, urine or cerebral spinal fluid in the case of 2-methylcitric acid, it should be appreciated that "serum” may include plasma and that it may be feasible to apply the assays to other body fluids as well.
  • FIG. 1 shows a diagram of the metabolic pathways of homocysteine, methionine and cystathionine in humans.
  • FIG. 2A and 2B illustrate the mass spectra of cystathionine.
  • FIG. 3A and 3B illustrates the chromatograms of cystathionine.
  • FIG. 4 shows a diagram of the metabolic pathways of 2-methylcitric acid and methylmalonic acid.
  • FIG. 5A and 5B illustrates the mass spectra of 2-methylcitric acid.
  • FIG. 6A and 6B illustrates the chromatograms of 2-methylcitric acid.
  • FIG. 7 shows clinical data showing the serum cystathionine levels in nanomoles per liter in normal patients and patients with cobalamin or folic acid deficiency.
  • FIG. 8 shows clinical data showing the 2-methylcitric acid levels in nanomoles per liter in normal patients and patients with cobalamin or folic acid deficiencies.
  • the metabolism of homocysteine, methionine and cystathionine in humans is generally known and is shown diagrammatically in FIG. 1.
  • Methionine is converted to S-adenosylmethionine by the transfer of the adenosyl moiety of ATP to methionine.
  • S-adenosylmethionine is a high energy compound, and each sulfonium atom is capable of participating in one or more transfer reactions to produce the sulfur-containing compound S-adenosylhomocysteine.
  • Hydrolysis cleaves S-adenosylhomocysteine into homocysteine and adenosine.
  • Homocysteine may be converted to cystathionine by the transsulfuration pathway or may be remethylated to form methionine.
  • the reaction utilizes N 5 -methyltetrahydrofolate (N 5 -MTHF) as the methyl donor and utilizes methylcobalamin as a cofactor.
  • N 5 -MTHF N 5 -methyltetrahydrofolate
  • the remethylation of homocysteine results in methionine and tetrahydrofolate, the later of which is converted back to N 5 -MTHF by DNA and RNA pathways.
  • a deficiency in either folic acid or cobalamin will block the methylation of homocysteine into methionine. Such a block may lead to increases in serum or urine levels of homocysteine and cystathionine, although this outcome is not necessarily apparent in view of the complexity of the metabolic pathways and the possibility of other reactions.
  • cystathionine In the synthesis of cystathionine from homocysteine in the transsulfuration pathway, the homocysteine is condensed with serine in a reaction catalyzed by cystathionine ⁇ synthase. Cystathionine cleaves into cysteine and ⁇ -ketobutyrate in a reaction catalyzed by ⁇ -cystathionase to complete the transsulfuration sequence.
  • propionyl-CoA is converted Into methylmalonyl-CoA.
  • the methylmalonyl is converted to succinyl-CoA in a reaction requiring adenosylcobalamin as a cofactor.
  • a deficiency in cobalamin may block the conversion of methylmalonyl-CoA to succinyl-CoA, thereby resulting in methylmalonic aciduria or propionic aciduria, although again such an outcome is not necessarily apparent from the metabolic pathway because a variety of other reactions could occur.
  • propionyl-CoA and oxaloacetic acid and water react to form 2-methylcitric acid, using the enzyme citrate synthase as a catalyst. It has been found that the blocking of the pathway from propionyl-CoA to succinyl-CoA by a deficiency of adenosylcobalamin may increase the conversion of propionyl-CoA to 2-methylcitric acid, thereby increasing 2-methylcitric acid levels.
  • cobalamin in the form of methylcobalamin
  • folic acid are vital to the methylation of homocysteine into methionine
  • cobalamin in the form of adenosylcobalamin but not folic acid is vital to the conversion of methylmalonyl-CoA to succinyl-CoA.
  • a deficiency in either cobalamin or folic acid may lead to increased levels of homocysteine or cystathionine.
  • a deficiency in cobalamin but not folic acid may also result in increased levels of methylmalonic acid or 2-methylcitric acid.
  • a preferred embodiment of the invention utilizes an assay for cystathionine levels in the manner described below.
  • the following components are added in sequence to 12 ⁇ 75 mm glass test tubes:
  • the samples are mixed well by vortexing and 51 ul of 0.083 H 3 BO 3 -NaOH, pH 10.0, containing 16.6 mg/ml of D, L-dithiothreitol is followed by mixing. After incubation of iodacctamide is added followed by mixing. After incubating for 30 minutes at 40° C., 1 ml of 0.03N HCl is added followed by mixing and the samples are then applied to 300 ul columns of a cation exchange resin (AG MP-50, 100-200 mesh, hydrogen form (Bio-Rad Laboratories, Richmond, Calif.)) that has previously been washed with 1 ml of MeOH and 3.3 ml of H 2 O, to remove any negatively charged salts.
  • a cation exchange resin AG MP-50, 100-200 mesh, hydrogen form (Bio-Rad Laboratories, Richmond, Calif.)
  • each column is washed twice with 3 ml of H 2 O and once with 3 ml of MeOH.
  • Each column is then eluted with 1.1 ml of 4N NH 4 OH in MeOH.
  • the eluates are taken to dryness by vacuum centrifugation in a Savant vacuum centrifuge.
  • the dried eluates are then derivatized by adding 30 ul of a solution containing 10 ul of N-methyl-N(tert-butyl dimethylsylyl) trifluoracetamide and 20 ul of acetonitrile. After incubation at 40° C.
  • Gas chromatography/mass spectrometry is performed using a Hewlett Packard 5890 Gas Chromatograph and a Hewlett Packard 5970 or 5971A Mass Selective Detector.
  • the initial column temperature is 140° C. which is held for approximately 0.6 minutes after sample injection and is then increased to 300° C. at a rate of 30° C./minute.
  • the mass selective detector is operated in the selected ion monitoring mode in which ions 362.2 are monitored for endogenous cystathionine and 366.2 for D,L[2,2,3,3-D 4 ] cystathionine.
  • Cystathionine is quantitated by dividing the integrated area of the M/Z 362.2 peak that elutes at approximately 5.7 minutes (the exact times are determined daily with controls) by the integrated area of the M/Z 366.2 peak that elutes at the same time and then multiplying by 1,000 nanomoles/liter, which is the equivalent amount of D,L[2,2,3,3-D 4 ] cystathionine that was added to each sample.
  • the method may be altered in the following manner: 1) 51 ul of H 2 O containing 400 picomoles of D,L[2,2,3,3-D 4 ] cystathionine, 400 ul of serum (or 40 ul of urine with 360 ul of 0.15M NaCl), and 1 ml of H 2 O are added in the initial sequence; 2) 51 ul of 1N NaOH containing 10 mg/ml of dithiothreitol are added in place of the H 3 BO 3 dithiothreitol prior to the first 30 minute incubation; 3) the addition of iodoacctamide and the second 30 minute incubation are omitted; 4) the 0.03N HCl is omitted and the samples are applied to 300 ul columns of an anion exchange resin (AG MP-1, 100-200 mesh, chloride form); and 5) samples are eluted with 0.04N acetic acid in methanol. Comparable results are obtained with both methods.
  • FIG. 2A and 2B show the molecular weights of cystathionine and a diagram of the cystathionine molecule. Peaks representing the entire derivative, i.e. [M] + , were not observed.
  • FIG. 3A and 3B shows the gas chromotogram of cystathionine, the top graph being for endogenous cystathionine and the bottom graph being for D,L[2,2,3,3-D 4 ] cystathionine.
  • a preferred embodiment of the invention utilizes an assay for 2-methylcitric acid levels in the manner described below.
  • the following components are each added in sequence to 12 ⁇ 75 mm glass test tubes:
  • the samples are mixed by vortexing and then applied to 300 ul columns of an anion exchange resin (AG MP-1, 100-200 mesh, chloride form (Bio-Rad Laboratories, Richmond, Calif.) that has previously been washed with 1 ml of MeOH and 3.3 ml of H 2 O. After the sample is applied, each column is washed with 3 ml of H 2 O and 3 times with 3 ml of 0.01N acidic acid in MeOH. Each column is then eluted with 1.1 ml of 3.6M acidic acid/0.1N HCl in MeOH. The eluates are taken to dryness by vacuum centrifugation in a Savant vacuum centrifuge.
  • an anion exchange resin AG MP-1, 100-200 mesh, chloride form (Bio-Rad Laboratories, Richmond, Calif.) that has previously been washed with 1 ml of MeOH and 3.3 ml of H 2 O. After the sample is applied, each column is washed with 3 ml of H 2 O
  • the dried eluates are then derivatized by adding 30 ul of a solution containing 10 ul of N-methyl-N(tert-butyl dimethylsylyl)trifluoracetamide and 20 ul of acetonitrile. After incubation at 90° C. for 30 minutes in sealed autosampler vials, 1 ul is analyzed by gas chromatography/mass spectrometry using a 20 meter SPB-1 capillary column (Supelco, Inc.) that has an internal diameter of 0.25 mm and a 0.25 um film thickness.
  • Gas chromatography/mass spectrometry is performed using a Hewlett Packard 5890 Gas Chromatograph and a Hewlett Packard 5971A Mass Selective Detector.
  • the initial column temperature is 80° C. which is held for approximately 0.6 minutes after sample injection and is then increased to 300° C. at a rate of 30° C./minute.
  • the mass selective detector is operated in the selected ion monitoring mode in which ions 605.4 are monitored for endogenous 2-methylcitric acid II and I and 608.4 are monitored for [methyl-D 3 ]2-methylcitric acid II and I.
  • 2-methylcitric acid II is quantitated by dividing the integrated area of the M/Z 605.4 peak that elutes at approximately 8.4 minutes (the exact times are determined daily with controls) by the integrated area of the M/Z 608.4 peak that elutes at the same time and then multiplying by 501 nanomoles/liter, which is the equivalent amount of [methyl-D 3 ]2-methylcitric acid II that was added to each sample.
  • 2-methylcitric acid I is quantitated in the same manner utilizing the M/Z 605.4 and M/Z 608.4 peaks that elute at approximately minutes and then multiplying their ratio by 432 nanomoles/liter which is the equivalent amount of [methyl-D 3 ]2-methylcitric acid I added to each sample.
  • the integrated areas for the two internal standard peaks i.e. the M/Z 608.4 peaks eluting at about 8.4 and 8.5 minutes, are corrected for the amounts contributed to them by endogenous 2-methylcitric acid II and I, as a result of naturally occurring isotope abundance.
  • These corrections which are determined using samples containing only unenriched 2-methylcitric acid II and I, are approximately 6.6% of the areas of the 605.4 peaks at 8.4 and 8.5 minutes. It has been found that some serum, urine and cerebral spinal fluid samples contain an endogenous peak of M/Z 608.4 that elutes at the same time, i.e.
  • the same process may be used to assay for methylmalonic acid if a suitable internal standard for it is added to the samples.
  • FIG. 5A and 5B show the molecular weights of 2-methylcitric acid and a diagram of the 2-methylcitric acid molecule. Peaks representing the entire molecule, i.e. [M] + were not observed.
  • FIG. 6A and 6B show the gas chromatogram of 2-methylcitric acid I and II, the top graph being for endogenous 2-methylcitric acid I and II and the bottom graph being for [methyl-D 3 ]2-methylcitric acid I and II.
  • the second method for assaying cystathionine was combined with the method for assaying 2-methylcitric acid in the following manner: 1) 51 ul of H 2 O containing the internal standards for cystathionine, 2-methylcitric acid, homocysteine, and methylmalonic acid was added to 400 ul of serum or cerebral spinal fluid or urine (40 ul urine plus 360 ul of 0.15M NaCl) and 1 ml of H 2 O in the initial sequence; 2) 51 ul of H 2 O containing 20 mg/ml of dithiothreitol was then added followed by, mixing and a 30 minute incubation at 40° C.; 3) the entire sample was then treated and followed up exactly as described for the 2-methylcitrate assay except that the run-through from the AG MP-1 was saved instead of being discarded; 4) 51 ul of 1N NaOH containing 10 mg/ml of dithiothreitol was added to the run-through followed by mixing and a second
  • FIG. 7 shows on a logarithmic scale the serum cystathionine levels in nanomoles per liter in 50 human patients having no manifestations of any cobalamin or folic acid deficiency, in 30 human patients having a known cobalamin deficiency and in 20 human patients having a known folic acid deficiency.
  • high serum levels of cystathionine generally but not always correspond to either cobalamin or folic acid deficiency.
  • Table II set forth below shows serum 2-methylcitric acid levels in nanomoles per liter for 50 patients with no cobalamin deficiency.
  • the table is broken into columns for total 2-methylcitric acid (“TOTMC”), the ratio of the two isomers of 2-methylcitric acid (“MCI/MCII”), the second isomer (“MCII”) and the first isomer (“MCI”).
  • Table III set forth below shows serum 2-methylcitric acid levels for 50 human patients with clinically confirmed cobalamin deficiencies, using the same units and column labels as in Table II.
  • Table IV set forth below shows serum 2-methylcitric acid levels for 25 human patients with clinically confirmed folic acid deficiencies, using the same units and column labels as in Table II.
  • Table V set forth below shows urine 2-methylcitric acid levels from the same normal subjects as presented in Table II for serum 2-methylcitric acid levels.
  • the table is broken into columns for total urine 2-methylcitric acid ("UMCTOT"), the ratio for the two isomers of urine 2-methylcitric acid (“UMCI/UMCII”), the second isomer (“UMCII”) and the first isomer (“UMCI”).
  • Table VI set forth below shows cerebral spinal fluid 2-methylcitric acid levels for 5 human patients with clinically confirmed cobalamin deficiencies, using the same units and column labels as in Table II. Samples 1-5 are from 5 different human patients, while samples 6 and 7 are from the same human patient as sample 5 during (sample 6) and after (sample 7) cobalamin therapy.
  • Table VII set forth below shows the declining levels of both serum and urine cystathionine and 2-methylcitric acid levels in a cobalamin deficient human patient that is periodically administered 1000 ug doses of cobalamin over a 13 day period. The cobalamin administrations took place on days 0, 2, 6 and 13
  • Table VIII set forth below shows the serum cystathionine and 2-methylcitric acid levels in nanomoles per liter in a cobalamin human patient that was mistakenly treated with oral folic acid at the rate of one mg per day from days 0-11, and then was treated with weekly cobalamin injections of 1000 ug starting on day 35.
  • the folic acid treatments had no significant effect in reducing cystathionine or 2-methylcitric acid levels, but the cobalamin treatment did decrease both cystathionine and 2-methylcitric acid to approximately normal levels.

Abstract

A method for screening and distinguishing between cobalamin deficiency and folic acid deficiency by relating elevated levels of cystathionine to cobalamin or folic acid deficiency and relating elevated levels of 2-methylcitric acid to cobalamin deficiency but not folic acid deficiency. The methods can be used alone or in combination with other methods for detecting and distinguishing between cobalamin deficiency and folic acid deficiency.

Description

RELATED APPLICATIONS
This application is a continuation-in-part of application Ser. No. 333,124 filed Apr. 3, 1989 now abandoned and application Ser. No. 345,885 filed May 1, 1989.
FIELD OF THE INVENTION
This invention relates to a method of diagnosing cobalamin deficiency and folic acid deficiency and distinguishing between the two, in warm-blooded animals, particularly humans, by measuring serum levels of cystathionine and 2-methylcitric acid.
BACKGROUND OF THE INVENTION
Accurate and early diagnosis of cobalamin (Vitamin B12) and folic acid deficiencies in warm-blooded animals is important because these deficiencies can lead to life-threatening hematologic abnormalities which are completely reversible by treatment with cobalamin or folic acid, respectively. Accurate and early diagnosis of cobalamin deficiency is especially important because it can also lead to incapacitating and life-threatening neuropsychiatric abnormalities; administration of exogenous cobalamin stops the progression of these abnormalities, almost always leads to significant improvement in symptoms, and frequently leads to their complete correction. The distinction between cobalamin and folic acid deficiency is often difficult because both deficiencies lead to indistinguishable hematologic abnormalities; the distinction is important because use of the proper vitamin results in the greatest improvement in hematologic abnormalities and, more importantly, only cobalamin will correct the neuropsychiatric abnormalities which are only seen in cobalamin deficiencies. The use of folic acid to treat cobalamin deficiency is extremely dangerous, since some or all of the hematologic abnormalities may improve, but neuropsychiatric abnormalities will not improve and may progress, or even be precipitated.
Assays for cobalamin and folate in serum or plasma have long been the most widely utilized and recommended tests for diagnosing and distinguishing cobalamin and folic acid deficiency. However, in 1978 it was discovered that cobalamin analogues are present in human plasma and that their presence could mask cobalamin deficiency because the radioisotope dilution assays for serum cobalamin then in use were not specific for true cobalamin. This problem could be corrected by using pure or purified intrinsic factor as the binding protein in the radioisotope dilution assay for cobalamin. This modification has almost totally replaced assays existing in 1978 that used a nonspecific cobalamin-binding protein. See, e.g., U.S. Pat. No. 4,188,189 (Allen), U.S. Pat. No. 4,351,822 (Allen), U.S. Pat. No. 4,451,571 (Allen), and Kolhouse, J. F., H. Kondo, N. C. Allen, E. Podell, and R. H. Allen, N. Eng. J. Med. 299:785-792 (1978). These improved assays for serum cobalamin are now utilized in thousands of laboratories throughout the world and appear to give low values for about 90% of patients with cobalamin deficiency. R. H. Allen, S. P. Stabler, D. G. Savage and J. Lindenbaum, American Journal of Hematology, 34:90-98 (1990); J. Lindenbaum, D. G. Savage, S. P. Stabler and R. H. Allen, American Journal of Hematology., 34:99-107 (1990); S. P. Stabler, R. H. Allen, D. G. Savage and J. Lindenbaum, Blood, 76(5): 871-81 (1990).
The improved assays avoid the problem of cobalamin analogues masking true cobalamin deficiency, but they have been severely criticized because they frequently give low values in patients who lack any evidence of actual cobalamin deficiency. This problem of false positive testing led experts in the field to take the position that cobalamin deficiency should be considered and serum cobalamin values should be obtained only in patients who have hematologic or neurologic abnormalities that are typical of patients with cobalamin deficiency. Dr. Schilling and his coworkers, who are experts in the field of cobalamin deficiency and laboratory diagnosis, stated:
"We conclude that the `improved` vitamin B12 assay kits will yield an increased proportion of clinically unexplained low results for serum B12.
It seems prudent for scientific and economic reasons to measure serum vitamin B12 only in patients who have hematological or neurological findings that suggest a reasonable probability of vitamin B12 deficiency. Measuring serum B12 as a screening test in the anemic or the geriatric population will result in a high proportion of low values that cannot be correlated with clinical disease."
Schilling, R. F., V. F. Fairbanks, R. Miller, K. Schmitt, and M. J. Smith, Clin. Chem. 29(3):582-583 (1983).
Thus, the cobalamin assays referred to by Dr. Schilling frequently provided low serum cobalamin levels in patients who were not truly cobalamin deficient. Such findings are confusing or misleading to the physician and may result in unnecessary and expensive further testing. To avoid that, it was generally taught in the art that the clinical spectrum of cobalamin deficiency is relatively narrow and well-defined and that the possibility of cobalamin deficiency should only be considered in those who have concurrent hematological or neurological symptoms. Routine screening of the general population or those with only moderate anemia, or moderate macrocytosis, or other neuropsychiatric abnormalities, would lead to high numbers of false positives.
It was thought that the hematological or neurological symptoms that were necessary to justify an assay for serum cobalamin and the resulting risk of a false positive, were fairly severe. Those symptoms included significant anemia, displayed for example in decreased hematocrit or hemoglobin, with macrocytic red blood cells (i.e., mean cell volume (MCV) generally greater than 100 fl), or neurologic symptoms of peripheral neuropathy and/or ataxia. Anemia associated with cobalamin deficiency was described as typically severe with hemoglobin ≦8 g % or hematocrit <25% and the size of the red blood cells was described as greatly increased to levels >110 fl. See, for example, Babior and Bunn (1983) in Harrison's Principles of Internal Medicine (Petersdorf et al., eds.) McGraw-Hill Book Co., New York; Lee and Gardner (1984) in Textbook of Family Practice, 3rd Ed. (Rakel, ed.) Saunders & Co, Philadelphia). While it was well recognized that individuals with cobalamin deficiency could display neurologic disorders in the absence of anemia, such situations were believed to be exceptional and rare. See Beck (1985) in Cecil Textbook of Medicine, 17th Ed. (Wyngaarden and Smith, eds.) W. B. Saunders, Philadelphia, p. 893-900; Babior and Bunn (1987) in Harrison's Principles of Internal Medicine, 11th Ed. (Braunwald et al., eds.) McGraw-Hill, New York pp. 1498-1504; Walton (1985) Brain's Diseases of the Nervous System, 9th Ed. Oxford University Press, Oxford, UK. The neurologic symptoms of cobalamin deficiency were considered to be late manifestations of the disease most typically occurring after the onset of anemia or, if they occurred first, were soon to be followed by the onset of anemia. See Woltmann (1919) Am. J. Med. Sci. 157:400-409; Victor and Lear (1956) Am. J. Med. 20:896-911.
It was later discovered that the clinical spectrum of cobalamin deficiency is much broader than previously recognized and that many cobalamin-deficient patients are not anemic, or only moderately anemic; that in many cases their red blood cells are not macrocytic, or only moderately macrocytic; that in many cases a variety of neurologic abnormalities other than peripheral neuropathy and ataxia are present; and that in many cases the serum cobalamin level is only slightly decreased and may actually be normal, even with the improved assays using purified intrinsic factor. See U.S. Pat. No. 4,940,658 by Allen et al; S. P. Stabler, R. H. Allen, D. G. Savage and J. Lindenbaum, Blood, 76(5): 871-81 (1990). Accordingly, there was a need for an improved assay for cobalamin deficiency, preferably one in which cobalamin deficiency could be distinguished from folic acid deficiency.
An improved assay is disclosed in U.S. Pat. No. 4,940,658, the contents of which are hereby incorporated by reference, by Allen et al., who are the inventors of the present invention. The Allen et al. patent teaches a method of assaying total homocysteine serum concentrations to predict cobalamin or folic acid deficiency, and assaying serum methylmalonic acid concentrations to distinguish between the two deficiencies. Briefly, it was determined that there were elevated levels of total homocysteine in about 85-95% of patients with cobalamin deficiency and in about 90-95% of patients with folic acid deficiency. Therefore, elevated homocysteine levels pointed toward one of those two deficiencies, but did not distinguish between the two. However, distinguishing between the two was assisted by the discovery that there were elevated levels of methylmalonic acid in about 85-95% of patients with cobalamin deficiency but methylmalonic acid levels were normal in patients with folic acid deficiency. The two assays are relatively inexpensive and accurate, and can be performed concurrently by taking a single serum sample.
The assay described in the Allen patent is a very important test procedure but may not be infallible in detecting either kind of deficiency or in distinguishing between the two. About 5-15% of the cobalamin deficiencies will not be detected because these patients will not show elevated homocysteine levels. About 5-10% of the folic acid deficiencies will not be detected because these patients will not show elevated homocysteine levels. When elevated homocysteine. Levels are in fact detected, thereby indicating either a cobalamin deficiency or folic acid deficiency, about 5-10% of the cobalamin deficiencies can not be distinguished from the folic acid deficiencies because the patients will not show elevated methylmalonic acid levels. Therefore the assays may produce a small but not insignificant number of false negative results.
The existence of this small potential of false negative results led the inventors of the present invention to develop another assay procedure to detect cobalamin and folic acid deficiencies and to distinguish between the two. It has been discovered that elevated levels of cystathionine are present in the serum and urine of about 80-90% of patients with cobalamin deficiency or folic acid deficiency. It has also been discovered that elevated levels of 2-methylcitric acid are present in the serum, urine and cerebral spinal fluid of 80-90% of patients with cobalamin deficiency but not in patients with folic acid deficiency. The present invention uses an assay for cystathionine as a test for the existence of either of the two deficiencies or as a check on an assay for total homocysteine when testing for the two deficiencies. It also uses an assay for 2-methylcitric acid as a test for distinguishing between the two deficiencies or as a check on an assay for methylmalonic acid in distinguishing between the two deficiencies.
It was previously known that increased amounts of cystathionine are present in the urine and serum of children and adults with inherited defects of cystathionase who cannot convert cystathionine to cysteine and alpha-ketobutyrate. The serum levels of cystathionine range from undetectable to as high as 80,000 nanomoles per liter. Serum levels of methionine, homocysteine and cysteine are usually not elevated in these patients. See, e.g., Mudd, S. H., H. L. Levy and F. Skovby, in The Metabolic Basis of Inherited Disease, 6th Ed. (C. R. Scriver, A. L. Beaudet, W. S. Sly and D. Valle, eds.) (McGraw-Hill, Inc., New York, 1989) pp. 693-734. Elevated levels of urine cystathionine have also been seen in a variety of other conditions including pyridoxine (Vitamin B6) deficiency, hyperthyroidism, liver disease, various tumors of the central nervous system and liver, and inherited defects of cystathionine transport in the kidney. See, e.g., Mudd, S. H., in The Metabolic Basis of Inherited Disease, supra.
Elevated levels of cystathionine are present in the urine and serum of some children with inherited defects involving methionine synthase, who cannot convert homocysteine and N5 -methyltetrahydrofolate to methionine and tetrahydrofolate, respectively. These patients usually have low levels of methionine, high levels of total homocysteine, and normal levels of cysteine in their serum. In these patients, the inherited defects were due to: 1) failure to synthesize N5 -methyltetrahydrofolate; 2) failure to synthesize methylcobalamin, which is a required co-factor for methionine synthase; and 3) a lack of the plasma transport protein transcobalamin II, which is required to deliver cobalamin to cells. See, e.g., Levy, H. L., S. H. Mudd, J. D. Schulman, P. M. Dryfus, and R. H. Abeles, Am. J. Med. 48:390-397, 197; Baumgartner, E. R., H. Wick, R. Mauere, N. Egli, and B. Steinmann, Helv. Paediat. Acta 34:.465-482, 1979; Ribes, A., M. A. Vilaseca, P. Briones, A. Maya; J. Sabater, P. Pascual, L. Alvarez, J. Ros and E. G. Pascual, J. Inher. Metabol. Dis. 7(Suppl. 2):129-130, 1984; Baumgartner, R., H. Wick, J. C. Linnell, E. Gaull, C. Bachmann, and B. Steinmann, Helv. Paediat. Acta. 34:483-496, 1979; Mudd, S. H., in The Metabolic Basis of Inherited Disease, supra; Carmel, R., A. A. Bedros, J. W. Mace, and S. I. Goodman, Blood 55:570-579, 1980.
On the other hand, other children with similar defects did not have elevated levels of cystathionine, which tended to cast doubt on the reliability of such an indicator. See, e.g., Goodman, S. I., P. G. Moe, K. B. Hammond, S. H. Mudd, and B. W. Uhlendorf, Biochem. Med 4:500-515, 1970; Barshop, B. A., J. Wolff, W. L. Nyhan, A. Yu, C. Pordanos, G. Jones, L. Sweetman, J. Leslie, J. Holm, R. Green, D. W. Jacobsen, B. A. Cooper, and D. Rosenblatt, Am. J. Med. Genet. 35:222-228, 1990; Baumgartner, R., H. Wick, H. C. Linnell, E Gaull, C. Bachmann, and B. Steinmann, Helv. Paediat. Acta. 34:483-496, 1979; Mudd, S. H., in The Metabolic Basis of Inherited Disease, supra.
Elevated levels of cystathionine have been reported in the serum of pigs with severe experimental vitamin B12 deficiency. See Levy, H. L. and G. J. Cardinale, Fed. Proc. 29:634, 1970: Mudd, S. H., in Heritable Disorders of Amino Acid Metabolism: Patterns of Clinical Expression and Genetic Variation, (W. O. Nyhan, ed.) John Wiley & Sons, New York, 1974, pp. 429-451. Increased amounts of cystathionine have been observed in the urine of several children with life-threatening cobalamin deficiency. See Higgenbottom, M. C., L. Sweetman, and W. L. Nyhan, N. Engl. J. Med. 299:317-323, 1978; Davis, Jr., J. R., J. Goldenring, and B. H. Lugin, Am. J. Dis. Child. 135:566-567, 1981. In other infants with life-threatening cobalamin deficiency, amino acids in urine were found to be normal or cystathionine was present in undetectable or normal amounts. See Grasbeck, R., R. Gordin, I. Kantero, and B. Kuhlback, Acta Medica Scandinavica. 167:289-296, 1960; Lambert, H. P., T. A. J. Prankerd, and J. M. Smellie, Q. J. Med. 30:71-92, 1961; Lampkin, B. C. and A. M. Mauer, Blood 30:495-502, 1967; Hollowell, Jr., J. G., W. K. Hall, M. E. Coryell, J. McPherson, Jr., and D. A. Hahn, Lancet 2:1428, 1969; Frader, J., B. Reibman, and D. Turkewitz, N. Engl. J. Med. 299:1319-1320, 1978. Cystathionine was not detected in the serum or urine of a child with life-threatening folic acid deficiency although it may have been present in this patient's cerebral spinal fluid. See Corbeel, L., G. Van den Berghe, J. Jaeken, J. Van Tornout, and R. Eeckels, Eur. J. Pediatr. 143:284-290, 1985. It is believed that elevated cystathionine has not been reported in the serum or urine of children with moderate or mild cobalamin or folic acid deficiencies, or in adults with any degree of cobalamin or folic acid deficiencies, or in the serum of normal children or adults.
It was previously known that large amounts of 2-methylcitric acid are present in the urine of children with inherited defects in propionyl-CoA carboxylase, who cannot convert propionyl-CoA to D-methylmalonyl-CoA, and with inherited defects in L-methylmalonyl-CoA mutase, who cannot convert L-methylmalonyl-CoA to succinyl-CoA. See, e.g., Ando, T., K. Rasmussen, J. M. Wright, and W. L. Nyhan, J. Biol. Chem. 247:2200-2204, 1972: Sweetman, L., W. Weyler, T. Shafai, P. E. Young, and W. L. Nyhan, JAMA 242:1048-1052, 1979; Weidman, S. W. and G. R. Drysdale, Biochem. J. 177:169-174, 1979. It has also been reported that 2-methylcitric acid is present in increased amounts in the amniotic fluid and urine of pregnant women with fetuses that were shown after birth to have inherited defects in either propionyl-CoA carboxylase or L-methylmalonyl-CoA mutase. See Naylor, G., L. Sweetman, W. L. Nyhan, C. Hornbeck, J. Griffiths, L. Morch, and S. Brandange, Clinica Chimica Acta 107:175-183, 1980; Sweetman, L., G. Naylor, T. Ladner, J. Holm, W. L. Nyhan, C. Hornbeck, J. Griffiths, L. Morch, S. Brandange, L. Gruenke, and J. C. Craig, in Stable Isotopes (H. L. Schmidt, H. Forstel, and K. Heinzinger, eds.) Elsevier Scientific Publishing Company, Amsterdam, 1982, pp. 287- 293; Aramaki, S., D. Lehotay, W. L. Nyahn, P.M. MacLeod and L. Sweetman, J. Inher. Metab. Dis. 12:86-88, 1989; Coude, M., B. Chadefaux, D. Rabier, and P. Kamound, Clinica Chimica Acta. 187:329-332, 1990.
Elevated levels of 2-methylcitric acid have also been observed in the urine of some (Barshop, B. A., J. Wolff, W. L. Nyhan, A. Yu, C. Prodanos, G. Jones, L. Sweetman, J. Leslie, J. Holm, R. Green, D. W. Jacobsen, B. A. Cooper, and D. Rosenblatt, Am. J. Med. Genet. 35:222-228, 1990; Baumgartner, R., H. Wick, J. C. Linnell, E. Gaull, C.Bachmann, B. Steimann, Helv. Paediat. Acta. 34:483-496, 1979; Higgenbottom, M. C., L. Sweetman, and W. L. Nyhan, N. England J. Med. 299:317-323, 1978) but not all (Levy, H. L., S. H. Mudd, J. D. Schulman, P. M. Dreyfus, and R. H. Abelese, Am. J. Med. 48:390-397, 1970; Baumgartner, E. R. H. Wick, R. Maurer, N. Egli, and B. Steinmann, Helv. Paediat. Acta. 34:465-482, 1979; Ribes, A., M. A. Vilaseca, P. Briones, A. Maya and J. Sabater, J. Inherit. Metabol. Dis. 7(Suppl. 2):129-130, 1984; Mudd, S. H., and B. W. Uhlendorf, Biochem. Med. 4:500-515, 1970; Carmel, R., A. A. Bedros, J. W. Mace, and S. I. Goodman, Blood 55:570-579, 1980) children with inherited defects involving: 1) the inability to convert cobalamin to adenosyl-cobalamin, which is a required co-factor for L-methylmalonyl-CoA mutase; and 2) transcobalamin II deficiency which results in the inability to transport cobalamin from plasma to cells.
Large amounts of 2-methylcitric acid have been found in the urine of one child with life-threatening cobalamin deficiency. See Higgenbottom, M. C. L. Sweetman, and W. L. Nyhan, N. Engl. J. Med. 299:317-323, 1978. In other infants with life-threatening cobalamin deficiency, the presence or elevated levels of 2-methylcitric acid were not observed. See Davis, Jr., J. R., J. Goldenring, and B. H. Lubin, Am. J. Dis. Child. 135:566-567, 1989; Hollowell, Jr. J. G., W. K. Hall, M. E. Coryell, J. McPherson, Jr., and D. A. Hahn, Lancet 2:1428, 1969; Frader, J., Reibman, and D. Turkewitz, N. Engl. J. Med. 299:1319-1320, 1978). It is believed that the presence of 2-methylcitric acid in detectable or elevated amounts has not been reported in the urine of children with moderate or mild cobalamin deficiency or adults with severe, moderate or mild cobalamin deficiency. The presence of 2-methylcitric acid in detectable or elevated amounts has not been reported in the serum of any subject with any of the inherited disorders involving propionyl-CoA carboxylase, L-methylmalonyl-CoA mutase, the synthesis of adenosyl cobalamin or the plasma transport of cobalamin nor has it been reported in the serum of children or adults with any degree of cobalamin deficiency nor in the serum of normal children or adults.
SUMMARY OF THE INVENTION
The present invention measures cystathionine levels in the serum or urine and measures 2-methylcitric acid levels in the serum, urine or cerebral spinal fluid for the purpose of predicting cobalamin and folic acid deficiencies and for distinguishing between the two deficiencies. It has been determined that elevated cystathionine levels tend to correspond with the existence of either such deficiency, while elevated 2-methylcitric acid levels tend to correspond with the existence of cobalamin deficiency but not folic acid deficiency and can thereby be used to distinguish between the two deficiencies.
In a preferred embodiment, samples of serum or urine in the assay for cystathionine, and samples of serum, urine or cerebral spinal fluid in the assay for 2-methylcitric acid, are mixed by vortexing with water and known amounts of stable isotope forms of cystathionine or 2-methylcitric acid, respectively. The mixtures are washed, incubated, eluted and dried in the manner described herein, and then are analyzed by gas chromatography/mass spectrometry.
The assays for cystathionine and 2-methylcitric acid may be combined with assays for homocysteine or methylmalonic acid or both. Further, while the assays are described for use with serum or urine in the case of cystathionine and with serum, urine or cerebral spinal fluid in the case of 2-methylcitric acid, it should be appreciated that "serum" may include plasma and that it may be feasible to apply the assays to other body fluids as well.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a diagram of the metabolic pathways of homocysteine, methionine and cystathionine in humans.
FIG. 2A and 2B illustrate the mass spectra of cystathionine.
FIG. 3A and 3B illustrates the chromatograms of cystathionine.
FIG. 4 shows a diagram of the metabolic pathways of 2-methylcitric acid and methylmalonic acid.
FIG. 5A and 5B illustrates the mass spectra of 2-methylcitric acid.
FIG. 6A and 6B illustrates the chromatograms of 2-methylcitric acid.
FIG. 7 shows clinical data showing the serum cystathionine levels in nanomoles per liter in normal patients and patients with cobalamin or folic acid deficiency.
FIG. 8 shows clinical data showing the 2-methylcitric acid levels in nanomoles per liter in normal patients and patients with cobalamin or folic acid deficiencies.
DETAILED DESCRIPTION OF THE INVENTION The Metabolism
The metabolism of homocysteine, methionine and cystathionine in humans is generally known and is shown diagrammatically in FIG. 1. Methionine is converted to S-adenosylmethionine by the transfer of the adenosyl moiety of ATP to methionine. S-adenosylmethionine is a high energy compound, and each sulfonium atom is capable of participating in one or more transfer reactions to produce the sulfur-containing compound S-adenosylhomocysteine. Hydrolysis cleaves S-adenosylhomocysteine into homocysteine and adenosine.
Homocysteine may be converted to cystathionine by the transsulfuration pathway or may be remethylated to form methionine. In methylation, the reaction utilizes N5 -methyltetrahydrofolate (N5 -MTHF) as the methyl donor and utilizes methylcobalamin as a cofactor. The remethylation of homocysteine results in methionine and tetrahydrofolate, the later of which is converted back to N5 -MTHF by DNA and RNA pathways. A deficiency in either folic acid or cobalamin will block the methylation of homocysteine into methionine. Such a block may lead to increases in serum or urine levels of homocysteine and cystathionine, although this outcome is not necessarily apparent in view of the complexity of the metabolic pathways and the possibility of other reactions.
In the synthesis of cystathionine from homocysteine in the transsulfuration pathway, the homocysteine is condensed with serine in a reaction catalyzed by cystathionine β synthase. Cystathionine cleaves into cysteine and α-ketobutyrate in a reaction catalyzed by γ-cystathionase to complete the transsulfuration sequence.
In the pathway for the formation of methylmalonic acid, shown in FIG. 4, propionyl-CoA is converted Into methylmalonyl-CoA. The methylmalonyl is converted to succinyl-CoA in a reaction requiring adenosylcobalamin as a cofactor. A deficiency in cobalamin may block the conversion of methylmalonyl-CoA to succinyl-CoA, thereby resulting in methylmalonic aciduria or propionic aciduria, although again such an outcome is not necessarily apparent from the metabolic pathway because a variety of other reactions could occur.
In the synthesis of 2-methylcitric acid, propionyl-CoA and oxaloacetic acid and water react to form 2-methylcitric acid, using the enzyme citrate synthase as a catalyst. It has been found that the blocking of the pathway from propionyl-CoA to succinyl-CoA by a deficiency of adenosylcobalamin may increase the conversion of propionyl-CoA to 2-methylcitric acid, thereby increasing 2-methylcitric acid levels.
Thus it can be seen that cobalamin (in the form of methylcobalamin) and folic acid are vital to the methylation of homocysteine into methionine, and that cobalamin (in the form of adenosylcobalamin) but not folic acid is vital to the conversion of methylmalonyl-CoA to succinyl-CoA. A deficiency in either cobalamin or folic acid may lead to increased levels of homocysteine or cystathionine. A deficiency in cobalamin but not folic acid may also result in increased levels of methylmalonic acid or 2-methylcitric acid.
Assay for Cystathionine
A preferred embodiment of the invention utilizes an assay for cystathionine levels in the manner described below. The following components are added in sequence to 12×75 mm glass test tubes:
(1) 51 ul of H2 O containing 40 picomoles of D, L [2,2,3,3-D4 ] cystathionine, available from MSD Isotopes, Montreal, Canada, as a custom synthesis;
(2) 40 ul of serum or urine; and
(3) 100 ul of H2 O.
The samples are mixed well by vortexing and 51 ul of 0.083 H3 BO3 -NaOH, pH 10.0, containing 16.6 mg/ml of D, L-dithiothreitol is followed by mixing. After incubation of iodacctamide is added followed by mixing. After incubating for 30 minutes at 40° C., 1 ml of 0.03N HCl is added followed by mixing and the samples are then applied to 300 ul columns of a cation exchange resin (AG MP-50, 100-200 mesh, hydrogen form (Bio-Rad Laboratories, Richmond, Calif.)) that has previously been washed with 1 ml of MeOH and 3.3 ml of H2 O, to remove any negatively charged salts. After the sample is applied, each column is washed twice with 3 ml of H2 O and once with 3 ml of MeOH. Each column is then eluted with 1.1 ml of 4N NH4 OH in MeOH. The eluates are taken to dryness by vacuum centrifugation in a Savant vacuum centrifuge. The dried eluates are then derivatized by adding 30 ul of a solution containing 10 ul of N-methyl-N(tert-butyl dimethylsylyl) trifluoracetamide and 20 ul of acetonitrile. After incubation at 40° C. for 60 minutes in sealed autosampler vials, 1 ul is analyzed by gas chromatography/mass spectrometry using a 10 meter SPB-1 capillary column (Supelco, Inc., Bellefonte, Pa.) that has an internal diameter of 0.25 mm and a 0.25 um film thickness.
Gas chromatography/mass spectrometry is performed using a Hewlett Packard 5890 Gas Chromatograph and a Hewlett Packard 5970 or 5971A Mass Selective Detector. The initial column temperature is 140° C. which is held for approximately 0.6 minutes after sample injection and is then increased to 300° C. at a rate of 30° C./minute. The mass selective detector is operated in the selected ion monitoring mode in which ions 362.2 are monitored for endogenous cystathionine and 366.2 for D,L[2,2,3,3-D4 ] cystathionine. Cystathionine is quantitated by dividing the integrated area of the M/Z 362.2 peak that elutes at approximately 5.7 minutes (the exact times are determined daily with controls) by the integrated area of the M/Z 366.2 peak that elutes at the same time and then multiplying by 1,000 nanomoles/liter, which is the equivalent amount of D,L[2,2,3,3-D4 ] cystathionine that was added to each sample.
In some experiments, the method may be altered in the following manner: 1) 51 ul of H2 O containing 400 picomoles of D,L[2,2,3,3-D4 ] cystathionine, 400 ul of serum (or 40 ul of urine with 360 ul of 0.15M NaCl), and 1 ml of H2 O are added in the initial sequence; 2) 51 ul of 1N NaOH containing 10 mg/ml of dithiothreitol are added in place of the H3 BO3 dithiothreitol prior to the first 30 minute incubation; 3) the addition of iodoacctamide and the second 30 minute incubation are omitted; 4) the 0.03N HCl is omitted and the samples are applied to 300 ul columns of an anion exchange resin (AG MP-1, 100-200 mesh, chloride form); and 5) samples are eluted with 0.04N acetic acid in methanol. Comparable results are obtained with both methods.
The same processes may be used to assay for total homocysteine if a suitable internal standard for added to the samples.
FIG. 2A and 2B show the molecular weights of cystathionine and a diagram of the cystathionine molecule. Peaks representing the entire derivative, i.e. [M]+, were not observed. FIG. 3A and 3B shows the gas chromotogram of cystathionine, the top graph being for endogenous cystathionine and the bottom graph being for D,L[2,2,3,3-D4 ] cystathionine.
Assay for 2-Methylcitric Acid
A preferred embodiment of the invention utilizes an assay for 2-methylcitric acid levels in the manner described below. The following components are each added in sequence to 12×75 mm glass test tubes:
1) 51 ul of H2 O containing 200.4 picomoles of [methyl-D3 ]2-methylcitric acid II and 172.8 picomoles of [methyl-D3 ]2-methylcitric acid I available from MSD Isotopes, Montreal, Canada, as a custom synthesis;
2) 400 ul of serum, cerebral spinal fluid, or urine (in the case of urine, 40 ul are used together with 360 ul of 0.15M NaCl); and
3) 1 ml of H2 O.
The samples are mixed by vortexing and then applied to 300 ul columns of an anion exchange resin (AG MP-1, 100-200 mesh, chloride form (Bio-Rad Laboratories, Richmond, Calif.) that has previously been washed with 1 ml of MeOH and 3.3 ml of H2 O. After the sample is applied, each column is washed with 3 ml of H2 O and 3 times with 3 ml of 0.01N acidic acid in MeOH. Each column is then eluted with 1.1 ml of 3.6M acidic acid/0.1N HCl in MeOH. The eluates are taken to dryness by vacuum centrifugation in a Savant vacuum centrifuge. The dried eluates are then derivatized by adding 30 ul of a solution containing 10 ul of N-methyl-N(tert-butyl dimethylsylyl)trifluoracetamide and 20 ul of acetonitrile. After incubation at 90° C. for 30 minutes in sealed autosampler vials, 1 ul is analyzed by gas chromatography/mass spectrometry using a 20 meter SPB-1 capillary column (Supelco, Inc.) that has an internal diameter of 0.25 mm and a 0.25 um film thickness.
Gas chromatography/mass spectrometry is performed using a Hewlett Packard 5890 Gas Chromatograph and a Hewlett Packard 5971A Mass Selective Detector. The initial column temperature is 80° C. which is held for approximately 0.6 minutes after sample injection and is then increased to 300° C. at a rate of 30° C./minute. The mass selective detector is operated in the selected ion monitoring mode in which ions 605.4 are monitored for endogenous 2-methylcitric acid II and I and 608.4 are monitored for [methyl-D3 ]2-methylcitric acid II and I. 2-methylcitric acid II is quantitated by dividing the integrated area of the M/Z 605.4 peak that elutes at approximately 8.4 minutes (the exact times are determined daily with controls) by the integrated area of the M/Z 608.4 peak that elutes at the same time and then multiplying by 501 nanomoles/liter, which is the equivalent amount of [methyl-D3 ]2-methylcitric acid II that was added to each sample. 2-methylcitric acid I is quantitated in the same manner utilizing the M/Z 605.4 and M/Z 608.4 peaks that elute at approximately minutes and then multiplying their ratio by 432 nanomoles/liter which is the equivalent amount of [methyl-D3 ]2-methylcitric acid I added to each sample. The integrated areas for the two internal standard peaks, i.e. the M/Z 608.4 peaks eluting at about 8.4 and 8.5 minutes, are corrected for the amounts contributed to them by endogenous 2-methylcitric acid II and I, as a result of naturally occurring isotope abundance. These corrections, which are determined using samples containing only unenriched 2-methylcitric acid II and I, are approximately 6.6% of the areas of the 605.4 peaks at 8.4 and 8.5 minutes. It has been found that some serum, urine and cerebral spinal fluid samples contain an endogenous peak of M/Z 608.4 that elutes at the same time, i.e. approximately 8.5 minutes, as the M/Z 608.4 peak for 2-methylcitric acid I and that this endogenous peak interferes with the quantitation of endogenous 2-methylcitric acid in these samples. This problem can be solved by using the M/Z 608.4 peak for 2-methylcitric acid II as the internal standard for quantitation of 2-methylcitric acid I and 2-methylcitric acid II.
The same process may be used to assay for methylmalonic acid if a suitable internal standard for it is added to the samples.
FIG. 5A and 5B show the molecular weights of 2-methylcitric acid and a diagram of the 2-methylcitric acid molecule. Peaks representing the entire molecule, i.e. [M]+ were not observed. FIG. 6A and 6B show the gas chromatogram of 2-methylcitric acid I and II, the top graph being for endogenous 2-methylcitric acid I and II and the bottom graph being for [methyl-D3 ]2-methylcitric acid I and II.
Although the assay described above is for both 2-methylcitric acid I and 2-methylcitric acid II, it should be appreciated that the assay can actually be used for either compound or both in combination.
Combined Assays
In other experiments of a preferred embodiment of the invention, the second method for assaying cystathionine was combined with the method for assaying 2-methylcitric acid in the following manner: 1) 51 ul of H2 O containing the internal standards for cystathionine, 2-methylcitric acid, homocysteine, and methylmalonic acid was added to 400 ul of serum or cerebral spinal fluid or urine (40 ul urine plus 360 ul of 0.15M NaCl) and 1 ml of H2 O in the initial sequence; 2) 51 ul of H2 O containing 20 mg/ml of dithiothreitol was then added followed by, mixing and a 30 minute incubation at 40° C.; 3) the entire sample was then treated and followed up exactly as described for the 2-methylcitrate assay except that the run-through from the AG MP-1 was saved instead of being discarded; 4) 51 ul of 1N NaOH containing 10 mg/ml of dithiothreitol was added to the run-through followed by mixing and a second 30 minute incubation at 40° C.; and 5) the treated run-through samples from the preceding step were then applied directly to AG MP-1 columns and eluted with 0.04N acetic acid and methanol exactly as described in the second cystathionine method. In this combined assay, two autosampler vials were obtained for each sample, with the first autosampler vial containing endogenous and internal standards for 2-methylcitric acid and methylmalonic acid and the second autosampler vial containing endogenous and internal standards for cystathionine and homocysteine. During the GC/MS analysis, the peaks for endogenous an the internal standard for methylmalonic acid eluted at approximately 5.0 minutes which were well separated and ahead of the corresponding peaks for 2-methylcitric acid which eluted at approximately 8.5 minutes (see above). During the GC/MS analysis, the peaks for endogenous and the internal standard for homocysteine eluted at approximately 3.8 minutes which were well separated and ahead of the corresponding peaks for cystathionine which eluted at approximately 5.7 minutes (see above).
Clinical Results
FIG. 7 shows on a logarithmic scale the serum cystathionine levels in nanomoles per liter in 50 human patients having no manifestations of any cobalamin or folic acid deficiency, in 30 human patients having a known cobalamin deficiency and in 20 human patients having a known folic acid deficiency. As the Figure suggests, high serum levels of cystathionine generally but not always correspond to either cobalamin or folic acid deficiency.
Table I set forth below shows urine cystathionine levels ("UCYSTAT") from 50 normal subjects in nanomoles per liter.
              TABLE I                                                     
______________________________________                                    
(normal urine cystathionine)                                              
SAMPLE ID          UCYSTAT                                                
______________________________________                                    
 1          NN01       7426                                               
 2          NN02       467                                                
 3          NN03       10021                                              
 4          NN04       6077                                               
 5          NN05       3887                                               
 6          NN06       13259                                              
 7          NN07       2343                                               
 8          NN08       2472                                               
 9          NN09       4817                                               
10          NN10       3736                                               
11          NN11       8945                                               
12          NN12       4144                                               
13          NN13       3515                                               
14          NN16       38834                                              
15          NN17       8329                                               
16          NN19       5723                                               
17          NN20       20761                                              
18          NN21       3594                                               
19          NN22       3603                                               
20          NN24       19063                                              
21          NN25       4515                                               
22          NN26       7361                                               
23          NN30       6665                                               
24          NN31       680                                                
25          NN32       2339                                               
26          NN33       6017                                               
27          NN34       3592                                               
28          NN35       547                                                
29          NN36       21236                                              
30          NN37       6811                                               
31          NN39       3245                                               
32          NN40       3474                                               
33          NN41       2632                                               
34          NN43       58                                                 
35          NN44       11953                                              
36          NN45       691                                                
37          NN46       5783                                               
38          NN47       3844                                               
39          NN48       1875                                               
40          NN49       3639                                               
41          NN50       877                                                
42          NN52       1599                                               
43          NN53       5318                                               
44          NNS4       1607                                               
45          NN55       2079                                               
46          NN56       936                                                
47          NN57       10747                                              
48          NN58       12060                                              
49          NN59       6175                                               
50          NN60       1644                                               
______________________________________
Table II set forth below shows serum 2-methylcitric acid levels in nanomoles per liter for 50 patients with no cobalamin deficiency. The table is broken into columns for total 2-methylcitric acid ("TOTMC"), the ratio of the two isomers of 2-methylcitric acid ("MCI/MCII"), the second isomer ("MCII") and the first isomer ("MCI").
              TABLE II                                                    
______________________________________                                    
(normal serum 2-methylcitric acid)                                        
SAMPLE ID  TOTMC    MCI/MCII    MCII  MCI                                 
______________________________________                                    
 1    NN55     282      .55       181   100                               
 2    NN03     228      .60       143   86                                
 3    NN46     216      .86       117   100                               
 4    NN11     211      .66       127   84                                
 5    NN06     195      .57       124   71                                
 6    NN18     186      .80       104   83                                
 7    NN58     176      .92       92    84                                
 8    NN49     174      .64       106   68                                
 9    NN25     171      .79       96    76                                
10    NN50     145      .66       87    58                                
11    NN26     138      .58       87    50                                
12    NN43     137      1.00      68    68                                
13    NN52     136      .65       83    54                                
14    NN35     136      1.09      65    71                                
15    NN07     131      .59       82    49                                
16    NN29     131      .59       82    49                                
17    NN57     128      .56       82    46                                
18    NN34     127      .75       73    55                                
19    NN13     126      .79       70    56                                
20    NN10     125      .58       79    46                                
21    NN20     125      .56       80    45                                
22    NN01     121      .55       78    43                                
23    NN22     121      .57       77    44                                
24    NN08     115      .64       70    45                                
25    NN04     115      .77       65    50                                
26    NN41     114      .52       75    39                                
27    NN19     114      .61       71    43                                
28    NN32     114      .58       72    42                                
29    NN39     110      .52       72    38                                
30    NN37     109      .75       62    47                                
31    NN24     109      .60       68    41                                
32    NN28     106      .66       64    42                                
33    NN21     100      .88       53    47                                
34    NN47     100      .53       65    35                                
35    NN33      94      .54       61    33                                
36    NN45      93      .62       58    36                                
37    NN02      93      .50       62    31                                
38    NN53      92      .48       62    30                                
39    NN05      92      .57       59    33                                
40    NN54      91      .95       47    45                                
41    NN16      91      .64       55    35                                
42    NN09      87      .54       57    31                                
43    NN31      83      .67       51    31                                
44    NN36      80      .58       51    29                                
45    NN56      79      .51       52    26                                
46    NN40      78      .54       50    27                                
47    NN48      70      .71       41    29                                
48    NN44      69      .74       40    29                                
49    NN17      66      .50       44    22                                
50    NN12      66      .77       37    28                                
______________________________________
Table III set forth below shows serum 2-methylcitric acid levels for 50 human patients with clinically confirmed cobalamin deficiencies, using the same units and column labels as in Table II.
              TABLE III                                                   
______________________________________                                    
(cobalamin deficient serum 2-methylcitric acid)                           
SAMPLE ID  MCTOT    MCI/MCII    MCII  MCI                                 
______________________________________                                    
 1    B7687    13509    .48       9121  4388                              
 2    12416    8826     .72       5146  3680                              
 3    B4942    6028     .49       4050  1978                              
 4    D3648    3296     .60       2057  1239                              
 5    A3172    3071     .72       1787  1284                              
 6    B2380    2965     .54       1924  1041                              
 7    C8881    2757     .59       1732  1025                              
 8    C9246    2728     .57       1739  989                               
 9    D6354    2676     .59       1688  988                               
10    D4205    2665     1.16      1232  1433                              
11    F1247    2594     .56       1663  930                               
12    B1330    2488     .62       1535  953                               
13    09267    2439     .54       1587  852                               
14    E1309    2251     .62       1385  865                               
15    C3384    2236     .64       1367  869                               
16    F1111    2230     .67       1334  896                               
17    F7167    1892     .56       1215  677                               
18    A3511    1731     .68       1029  702                               
19    C5237    1652     .68        986  666                               
20    C9834    1626     .65        986  640                               
21    X1079    1619     .50       1079  540                               
22    D0883a   1593     .58       1007  587                               
23    D7154    1482     .68        883  599                               
24    D2459    1411     .67        844  567                               
25    A8811    1345     .85        726  618                               
26    E8989    1244     .63        762  485                               
27    C9199    1236     .98        624  612                               
28    E9773    1068     .78        600  468                               
29    D3735    875      .58        553  322                               
30    F3628    791      .62        488  303                               
31    F2567    774      .56        497  276                               
32    F4201    697      .68        414  283                               
33    D1206    637      .83        347  289                               
34    D1321a   604      .68        359  245                               
35    C8686a   581      .66        350  231                               
36    D8953    483      .66        292  191                               
37    A5038    447      .85        242  206                               
38    C8800    398      .69        236  162                               
39    F9991    393      .69        233  160                               
40    D2361    370      .66        223  148                               
41    D2088    326      .66        197  130                               
42    C8227    300      .67        180  120                               
43    E0219    297      .58        188  109                               
44    12473    266      .66        160  106                               
45    D1397a   224      .69        132   92                               
46    D8330    214      .70        126   88                               
47    D4164    206      .50        137   69                               
48    C8872    203      .81        112   91                               
49    A2375    202      .54        131   71                               
50    F3977     93      .70        55    38                               
______________________________________
Table IV set forth below shows serum 2-methylcitric acid levels for 25 human patients with clinically confirmed folic acid deficiencies, using the same units and column labels as in Table II.
              TABLE IV                                                    
______________________________________                                    
(folic acid deficient 2-methylcitric acid)                                
SAMPLE ID  MCTOT    MCI/MCII    MCII  MCI                                 
______________________________________                                    
 1    D7348    247      1.46      100   147                               
 2    A7708    238      .83       130   108                               
 3    B9076    234      .50       156   78                                
 4    B7161    230      .66       139   92                                
 5    B7149    210      .49       141   69                                
 6    A8119    209      .59       132   77                                
 7    E9670    189      .73       109   80                                
 8    C5048    171      .76       97    74                                
 9    07601    166      .51       110   56                                
10    E6135    130      .54       84    46                                
11    11145    125      .90       66    59                                
12    C9169    123      1.07      60    64                                
13    E4663    105      .58       66    39                                
14    A7398a    94      .50       63    31                                
15    D0060     89      1.13      42    47                                
16    A3285a    66      .62       41    25                                
17    E9405     59      .75       34    25                                
18    E5653     52      .57       33    19                                
19    A3769b    48      .44       33    15                                
20    B4551     41      .52       27    14                                
21    A6199     40      .36       29    11                                
22    B6301     34      .50       22    11                                
23    F9010     26      .64       16    10                                
24    E4492     20      .51       13     7                                
25    D4162     10      .27        8     2                                
______________________________________
The data tabulated in Tables II-IV is presented in graphic form in FIG. 8 for the two 2-methylcitric acid isomers totalled. As evident from the tables and graph, high levels of 2-methylcitric acid tend to correspond to a cobalamin deficiency but not to a folic acid deficiency.
Table V set forth below shows urine 2-methylcitric acid levels from the same normal subjects as presented in Table II for serum 2-methylcitric acid levels. The table is broken into columns for total urine 2-methylcitric acid ("UMCTOT"), the ratio for the two isomers of urine 2-methylcitric acid ("UMCI/UMCII"), the second isomer ("UMCII") and the first isomer ("UMCI").
              TABLE V                                                     
______________________________________                                    
(normal urine 2-methylcitric acid)                                        
SAMPLE                                                                    
ID      UMCTOT    UMCI/UMCII   UMCII  UMCI                                
______________________________________                                    
 1  NN29    32296     .61        20101  12195                             
 2  NN26    26298     .58        16631  9667                              
 3  NN11    23574     .73        13608  9966                              
 4  NN06    22666     .65        13724  8941                              
 5  NN03    21617     .61        13429  8188                              
 6  NN21    20501     .83        11229  9272                              
 7  NN12    19289     .58        12225  7064                              
 8  NN48    19239     .58        12187  7052                              
 9  NN19    18999     .61        11797  7202                              
10  NN47    18651     .56        11962  6689                              
11  NN24    18279     .63        11220  7059                              
12  NN05    18141     .50        12075  6066                              
13  NN16    16259     .79         9491  6768                              
14  NN33    16227     .60        10143  6084                              
15  NN04    15363     .40        10996  4367                              
16  NN57    15238     .62         9402  5836                              
17  NN36    14761     .59         9293  5468                              
18  NN01    14463     .59         9098  5366                              
19  NN58    14243     .73         8223  6020                              
20  NN17    13851     .57         8840  5011                              
21  NN28    13492     .57         8611  4882                              
22  NN22    13395     .60         8346  5048                              
23  NN10    12630     .60         7914  4716                              
24  NN13    12339     .75         7044  5295                              
25  NN46    11740     .51          7755 3985                              
26  NN20    11527     .57         7328  4199                              
27  NN09    11094     .67         6647  4447                              
28  NN41    10893     .53         7140  3753                              
29  NN25     9883     .76         5620  4263                              
30  NN37     9330     .56         5995  3335                              
31  NN18     9200     .84         4999  4200                              
32  NN55     9165     .55         5908  3257                              
33  NN44     9162     .76         5203  3959                              
34  NN40     5910     .53         5807  3103                              
35  NN49     8246     .45         5687  2559                              
36  NN39     7733     .54         5014  2718                              
37  NN32     7358     .54         4778  2580                              
38  NN54     7159     .95         3672  3486                              
39  NN53     7145     .54         4643  2502                              
40  NN08     6265     .53         4100  2165                              
41  NN07     6008     .53         3935  2073                              
42  NN34     5570     .69         3303  2266                              
43  NN56     3492     .60         2180  1312                              
44  NN52     3416     .55         2204  1212                              
45  NN02     3062     .52         2018  1044                              
46  NN50     2535     .62         1564   972                              
47  NN35     2483     1.09        1185  1297                              
48  NN43     1631     .96         834    797                              
49  NN45     1617     .51         1068   549                              
50  NN31     859      .57         549    310                              
______________________________________
Table VI set forth below shows cerebral spinal fluid 2-methylcitric acid levels for 5 human patients with clinically confirmed cobalamin deficiencies, using the same units and column labels as in Table II. Samples 1-5 are from 5 different human patients, while samples 6 and 7 are from the same human patient as sample 5 during (sample 6) and after (sample 7) cobalamin therapy.
              TABLE VI                                                    
______________________________________                                    
(cobalamin deficient csf 2-methylcitric acid)                             
SAMPLE ID  TOTMC    MCI/MCII    MCII  MCI                                 
______________________________________                                    
 1    E9575    3742     1.71      1380  2363                              
 2    F2217    3140     3.43       709  2430                              
 3    Z0048    16238    1.86      5681  10557                             
 4    E9832    3967     3.49       884  3084                              
 5    F1033    5749     1.63      2184  3564                              
 6    F1420a   3660     2.13      1168  2492                              
 7    F2124     402     2.52       114   287                              
______________________________________
Table VII set forth below shows the declining levels of both serum and urine cystathionine and 2-methylcitric acid levels in a cobalamin deficient human patient that is periodically administered 1000 ug doses of cobalamin over a 13 day period. The cobalamin administrations took place on days 0, 2, 6 and 13
              TABLE VII                                                   
______________________________________                                    
                        MCI/                                              
TYPE SAMPLE ID                                                            
              TOTMC     MCII    MCII  MCI                                 
______________________________________                                    
 1  SERUM    DAY -1   1330    .69    786  544                             
 2  SERUM    DAY 0    963     .69    569  394                             
 3  SERUM    DAY 1    819     .63    502  317                             
 4  SERUM    DAY 2    814     .69    481  333                             
 5  SERUM    DAY 3    712     .69    420  292                             
 6  SERUM    DAY 6    347     .74    200  147                             
 7  SERUM    DAY 13   443     .54    287  156                             
 8  URINE    DAY -1   153709  .74   88404 65305                           
 9  URINE    DAY 0    120317  .72   69911 50406                           
10  URINE    DAY 1    146227  .69   86511 59716                           
11  URINE    DAY 2    65518   .71   38282 27236                           
12  URINE    DAY 3    76122   .71   44453 31670                           
13  URINE    DAY 6    30795   .73   17763 13031                           
14  URINE    DAY 13   17206   .59   10798 6408                            
______________________________________
Table VIII set forth below shows the serum cystathionine and 2-methylcitric acid levels in nanomoles per liter in a cobalamin human patient that was mistakenly treated with oral folic acid at the rate of one mg per day from days 0-11, and then was treated with weekly cobalamin injections of 1000 ug starting on day 35. As the table shows, the folic acid treatments had no significant effect in reducing cystathionine or 2-methylcitric acid levels, but the cobalamin treatment did decrease both cystathionine and 2-methylcitric acid to approximately normal levels.
              TABLE VIII                                                  
______________________________________                                    
DAY      CYSTA    MCTOTAL      MCII  MCI                                  
______________________________________                                    
 0       552      1242         709   533                                  
23       565       995         529   466                                  
26       513      1915         1128  787                                  
56       180       211         107   104                                  
______________________________________
From this clinical data, it can be concluded that elevated levels of serum or urine cystathionine levels suggest either a cobalamin deficiency or a folic acid deficiency. Further, elevated levels of serum, urine or cerebral spinal fluid 2-methylcitric acid suggest a cobalamin deficiency but not a folic acid deficiency. Once a cobalamin or folic acid deficiency is detected and distinguished, it can be effectively treated with administrations of the deficient compound. A significant decrease or normalization indicates that the deficiency was due to the vitamin used for treatment. The method described herein for detecting and distinguishing between cobalamin and folic acid deficiencies can be used alone or can be used in combination with or as a backup to other methods such as those that rely on measuring homocysteine or methylmalonic acid.

Claims (69)

What is claimed is:
1. A method for detecting a deficiency of cobalamin or folic acid in warm-blooded animals, comprising the steps of:
assaying a body fluid for an elevated level of cystathionine; and
correlating an elevated level of cystathionine in said body fluid with a likelihood of a deficiency of cobalamin or folic acid.
2. The method of claim 1, wherein elevated levels of cystathionine indicate a likelihood of a cobalamin or folic acid deficiency.
3. The method of claim 1, wherein said body fluid is one of serum and urine.
4. The method of claim 1, wherein the step of assaying for an elevated level of cystathionine includes:
combining said body fluid with a compound having cystathionine labelled with an isotope marker;
measuring the ratio of concentration of the labelled cystathionine and body fluid cystathionine present with a mass spectrometer; and
determining the concentration of body fluid cystathionine present in said body fluid.
5. The method of claim 4, wherein said step of assaying for an elevated level of cystathionine includes derivatizing the cystathionine before measuring the ratio of concentration of the labelled cystathionine and body fluid cystathionine.
6. The method of claim 5, wherein said derivatization is accomplished by exposing the cystathionine to N-methyl-N(tert-butyl dimethylsylyl) trifluoroacetamide.
7. The method of claim 4, wherein said compound includes deuterated cystathionine.
8. A method of diagnosing and treating a deficiency of cobalamin in a human patient, comprising detecting a cobalamin deficiency in accordance with the steps of claim 1, and administering cobalamin to the human patient in an amount sufficient to return the cystathionine levels to normal.
9. A method of for detecting a deficiency of cobalamin in warm-blooded animals, comprising the steps of:
assaying a body fluid for an elevated level of 2-methylcitric acid I or 2-methylcitric acid II or both; and
correlating an elevated level of 2-methylcitric acid I or 2-methylcitric acid II or both in said body fluid with a likelihood of a deficiency of cobalamin.
10. The method of claim 9, wherein elevated levels of 2-methylcitric acid I or 2-methylcitric acid II or both indicate a likelihood of a cobalamin deficiency.
11. The method of claim 9, wherein said body fluid is serum, urine or cerebral spinal fluid.
12. The method of claim 11, wherein said body fluid is cerebral spinal fluid.
13. The method of claim 9, wherein the step of assaying for an elevated level of 2-methylcitric acid I or 2-methylcitric acid II or both includes:
combining said body fluid with a compound having 2-methylcitric acid I labelled with an isotope marker or a compound having 2-methylcitric acid II labelled with an isotope marker or both;
measuring the ratio of concentration of the labelled 2-methylcitric acid I or 2-methylcitric acid II or both and body fluid 2-methylcitric acid I or 2-methylcitric acid II or both with a mass spectrometer; and
determining the concentration of body fluid 2-methylcitric acid I or 2-methylcitric acid II or both in said body fluid.
14. The method of claim 13, wherein said step of assaying for an elevated level of 2-methylcitric acid I or 2-methylcitric acid II or both includes derivatizing the 2-methylcitric acid I or 2-methylcitric acid II or both before measuring the ratio of concentration of the labelled 2-methylcitric acid I or 2-methylcitric acid II or both and body fluid 2-methylcitric acid I or 2-methylcitric acid II or both.
15. The method of claim 14, wherein said derivatization is accomplished by exposing the 2-methylcitric acid I or 2-methylcitric acid II or both to N-methyl-N(tert-butyl dimethylsylyl) trifluoroacetamide.
16. The method of claim 13, wherein said compound includes deuterated 2-methylcitric acid I or 2-methylcitric acid II or both.
17. A method of diagnosing and treating a deficiency of cobalamin in a human patient, comprising detecting a cobalamin deficiency in accordance with the steps of claim 9, and administering cobalamin to the human patient in an amount sufficient to return the levels of 2-methylcitric acid I or 2-methylcitric acid II or both to normal.
18. A method for detecting a deficiency of cobalamin or folic acid in warm-blooded animals and for distinguishing between a deficiency of cobalamin and a deficiency of folic acid, comprising the steps of:
assaying a first body fluid from said warm-blooded animal for an elevated level of cystathionine;
correlating an elevated level of cystathionine in said body fluid with a likelihood of a deficiency of cobalamin or folic acid;
assaying a second body fluid from said warm-blooded animal having an elevated level of cystathionine in said first body fluid correlating with a likelihood of a deficiency of cobalamin or folic acid, for an elevated level of 2-methylcitric acid I or 2-methylcitric acid II or both; and
correlating an elevated level of 2-methylcitric acid I or 2-methylcitric acid II or both in said second body fluid with a likelihood of a deficiency of cobalamin but both a likelihood of a deficiency of folic acid.
19. The method of claim 18, wherein elevated levels of cystathionine indicate a likelihood of a cobalamin of folic acid deficiency, and elevated levels of 2-methylcitric acid I or 2-methylcitric acid II or both indicate a likelihood of a cobalamin deficiency but not a likelihood of a folic acid deficiency.
20. The method of claim 19, wherein said first body fluid is serum or urine, and said second body fluid is serum, urine or cerebral spinal fluid.
21. The method of claim 20, wherein said first and second body fluids are the same.
22. The method of claim 18:
wherein the step of assaying for an elevated level of cystathionine includes combining said first body fluid with a first compound having cystathionine labelled with an isotope marker, and the step of assaying for an elevated level of 2- methylcitric acid I or 2-methylcitric acid II or both includes combining said second body fluid with a second compound having 2-methylcitric acid I or 2-methylcitric acid II or both labelled with an isotope marker; and
further comprising measuring the ratio of concentration of the labelled cystathionine and body fluid cystathionine and the ratio of concentration of the labelled 2-methylcitric acid I or 2-methylcitric acid II or both and body fluid 2-methylcitric acid I or 2-methylcitric acid II or both; and determining the concentration of body fluid cystathionine present in said first body fluid and determining the concentration of body fluid 2-methylcitric acid I or 2-methylcitric acid II or both in said second body fluid.
23. The method of claim 22, wherein said first and second body fluids are the same, and wherein the first compound having a known amount of labelled cystathionine and the second compound having a known amount of labelled 2-methylcitric acid I or 2-methylcitric acid II or both are added to a single sample or said body fluid, and wherein the single sample is divided into a first sample for measuring cystathionine and a second sample for measuring 2-methylcitric acid I or 2-methylcitric acid II or both.
24. The method of claim 22, wherein said steps of assaying for an elevated level of cystathionine and 2-methylcitric acid I or 2-methylcitric acid II or both includes derivatizing the cystathionine and 2-methylcitric acid I or 2-methylcitric acid II or both before measuring the ratio of concentration of the labelled cystathionine and body fluid cystathionine and the ratio of concentration of the labelled 2-methylcitric acid I or 2-methylcitric acid II or both and body fluid 2-methylcitric acid I or 2-methylcitric acid II or both.
25. The method of claim 24, wherein said derivatization is accomplished by exposing the cystathionine and 2-methylcitric acid I or 2-methylcitric acid II or both to N-methyl-N(tert-butyl dimethylsylyl)trifluoroacetamide.
26. The method of claim 22, wherein said first compound includes deuterated cystathionine and said second compound includes deuterated 2-methylcitric acid I of 2-methylcitric acid II or both.
27. The method of diagnosing and treating a deficiency of cobalamin in a human patient, comprising for detecting cobalamin deficiency in accordance with the steps of claim 18, and administering cobalamin to the human patient in an amount sufficient to return to normal the elevated level of cystathionine, or 2-methylcitric acid I or 2-methylcitric acid II or both.
28. A method of diagnosing and treating a deficiency of folic acid in a human patient, comprising detecting folic acid deficiency in accordance with the steps of claim 18, and administering folic acid to the human patient in an amount sufficient to return to normal the elevated level of cystathionine.
29. A method for detecting a deficiency of cobalamin or folic acid in warm-blooded animals, comprising the steps of:
assaying a first body fluid for an elevated level of cystathionine;
assaying a second body fluid for an elevated level of homocysteine; and
correlating an elevated level of cystathionine and homocysteine with a likelihood of a deficiency of cobalamin or folic acid.
30. The method of claim 29, wherein said first and second body fluids are serum or urine.
31. The method of claim 30, wherein said first and second body fluids are the same.
32. The method of claim 31, wherein said steps of assaying the body fluid for an elevated level of cystathionine and assaying said body fluid for an elevated level of homocysteine includes:
combining the body fluid with a first compound having a cystathionine labelled with an isotope marker and with a second compound having homocysteine labelled with an isotope marker;
measuring the ratio of concentration of the labelled cystathionine and body fluid cystathionine and the ratio of concentration of the labelled homocysteine and body fluid homocysteine with a mass spectrometer; and
determining the concentration of body fluid cystathionine and homocysteine present in said body fluid.
33. The method of claim 32, wherein the first compound and second compound are added to a single sample of said body fluid.
34. The method of claim 32, wherein said step of assaying for elevated levels of cystathionine and homocysteine includes derivatizing each of the cystathionine and homocysteine before measuring the ratio of concentration of each of the cystathionine and homocysteine and body fluid cystathionine and homocysteine.
35. The method of claim 34, wherein said derivatization is accomplished by exposing the cystathionine and homocysteine to N-methyl-N(tert-butyl dimethylsylyl)trifluoroacetamide.
36. The method of claim 32, wherein said first compound includes deuterated cystathionine and said second compound includes deuterated homocysteine.
37. The method of claim 28, further comprising the following steps for distinguishing between a likelihood of cobalamin deficiency and a likelihood of a folic acid deficiency:
assaying a third body fluid for an elevated level of at least one substance chosen from methylmalonic acid, 2-methylcitric acid I or 2-methylcitric acid II or both; and
correlating an elevated level of said chosen substance methylmalonic acid or 2-methylcitric acid I or 2-methylcitric acid II or both with a likelihood of a deficiency or cobalamin but not a likelihood of a deficiency of folic acid.
38. The method of claim 37, wherein the third body fluid is assayed for 2-methylcitric acid I or 2-methylcitric acid II or both and said body fluids are serum, urine or cerebral spinal fluid.
39. The method of claim 38, wherein said first, second and third body fluids are the same.
40. The method of claim 37, wherein the third body fluid is assayed for an elevated level of 2-methylcitric acid I or 2-methylcitric acid II or both in accordance with the steps of:
combining said third body fluid with a compound having 2-methylcitric acid I labelled with an isotope marker or 2-methylcitric acid II labelled with an isotope marker or both; measuring the ratio of concentration of labelled 2-methylcitric acid I or 2-methylcitric acid II or both and body fluid 2-methylcitric acid I or 2-methylcitric acid II or both with a mass spectrometer; and determining the concentration of body fluid 2-methylcitric acid I or 2-methylcitric acid II or both in said body fluid.
41. The method of claim 40, wherein said first, second and third body fluids are the same, and a first compound having labelled cystathionine, a second compound having labelled homocysteine and a third compound having labelled 2-methylcitric acid I or 2-methylcitric acid II or both are added to a single sample of said body fluid, and the single sample is divided into a first sample for measuring cystathionine and homocysteine and a second sample for measuring 2-methylcitric acid I or 2-methylcitric acid II or both.
42. The method of claim 37, wherein the third body fluid is assayed for 2-methylcitric acid I or 2-methylcitric acid II or both and said assay includes derivatizing the 2-methylcitric acid I or 2-methylcitric acid II or both before measuring the ratio of concentration of labelled 2-methylcitric acid I or 2-methylcitric acid II or both and body fluid 2-methylcitric acid I or 2-methylcitric acid II or both.
43. The method of claim 42, wherein said derivatization is accomplished by exposing the 2-methylcitric acid to N-methyl-N(tert-butyl dimethylsylyl) trifluoroacetamide.
44. The method of claim 40, wherein said third compound includes deuterated 2-methylcitric acid I or 2-methylcitric acid II or both.
45. A method for detecting a deficiency of cobalamin in warm-blooded animals, comprising the steps of:
assaying a first body fluid for elevated level of 2-methylcitric acid I or 2-methylcitric acid II or both;
assaying a second body fluid for an elevated level of methylmalonic acid; and
correlating an elevated level of 2-methylcitric acid I or 2-methylcitric acid II or both and methylmalonic acid with a likelihood of a deficiency of cobalamin.
46. The method of claim 45, wherein said first and second body fluids are serum, urine or cerebral spinal fluid.
47. The method of claim 46, wherein said first and second body fluids are the same.
48. The method of claim 45, wherein said step of assaying a first body fluid for an elevated level of 2-methylcitric acid I or 2-methylcitric acid II or both is in accordance with the steps of:
combining said third body fluid with a compound having 2-methylcitric acid I labelled with an isotope marker or 2-methylcitric acid II labelled with an isotope marker or both; measuring the ratio of concentration of labelled 2-methylcitric acid I or 2-methylcitric acid II or both and body fluid 2-methylcitric acid I or 2-methylcitric acid II or both with a mass spectrometer; and determining the concentration of body fluid 2-methylcitric acid I or 2-methylcitric acid II or both in said body fluid.
49. The method of claim 48, wherein said step of assaying a for an elevated level of 2-methylcitric acid I or 2-methylcitric acid II or both includes derivatizing the 2-methylcitric acid I or 2-methylcitric acid II or both before measuring the ratio of concentration of labelled 2-methylcitric acid I or 2-methylcitric acid II or both and the body fluid 2-methylcitric acid I or 2-methylcitric acid II or both.
50. The method of claim 49, wherein said derivatization is accomplished by exposing the 2-methylcitric acid I or 2-methylcitric acid II or both to N-methyl-N(tert-butyl dimethylsylyl) trifluoroacetamide.
51. The method of claim 48, wherein 2-methylcitric acid I or 2-methylcitric acid II or both includes deuterated 2-methylcitric acid I or 2-methylcitric acid II or both.
52. A method for detecting a deficiency of cobalamin or folic acid in warm-blooded animals, and for distinguishing between a deficiency of cobalamin and a deficiency of folic acid, comprising the steps of:
assaying a first body fluid for an elevated level of one of cystathionine and homocysteine;
correlating elevated levels of cystathionine and homocysteine with a likelihood of a deficiency of cobalamin or folic acid;
assaying a second body fluid for an elevated level of methylmalonic acid;
assaying a third body fluid for an elevated level of 2-methylcitric acid I or 2-methylcitric acid II or both; and
correlating elevated levels of methylmalonic acid and 2-methylcitric acid I or 2-methylcitric acid II or both with a likelihood of a deficiency of cobalamin but not a likelihood of a deficiency of folic acid.
53. The method of claim 52, wherein said first body fluid is serum or urine and said second and third body fluids are serum, urine or cerebral spinal fluid.
54. The method of claim 53, wherein said first, second and third body fluids are the same.
55. The method of claim 52, wherein said steps of assaying the first body fluid for an elevated level of one of cystathionine and homocysteine includes:
combining said first body fluid with a compound having one of cystathionine and homocysteine labelled with an isotope marker;
measuring the ratio of concentration of one of labelled cystathionine and body fluid cystathionine with a mass spectrometer; and
determining the concentration of body fluid cystathionine or homocysteine present in said body fluid.
56. The method of claim 55, wherein said step of assaying said third body fluid for an elevated level of 2-methylcitric acid I or 2-methylcitric acid II or both includes the steps of:
combining said third body fluid with a compound having 2-methylcitric acid I labelled with an isotope marker or 2-methylcitric acid II labelled with an isotope marker or both; measuring the ratio of concentration of labelled 2-methylcitric acid I or 2-methylcitric acid II or both and body fluid 2-methylcitric acid I or 2-methylcitric acid II or both with a mass spectrometer; and determining the concentration of body fluid 2-methylcitric acid I or 2-methylcitric acid II or both in said body fluid.
57. The method of claim 56, wherein said first, second and third body fluids are the same, and a first compound having one of cystathionine and homocysteine, a second compound having labelled methylmalonic acid, and a third compound having labelled 2-methylcitric acid I or 2-methylcitric acid II or both are added to a single sample of said body fluid, and the single sample is divided into a first sample for measuring said one of cystathionine and homocysteine and a second sample for measuring methylmalonic acid and 2-methylcitric acid I or 2-methylcitric acid II or both.
58. The method of claim 56, wherein said steps of assaying includes derivatizing the assayed compound before measuring the ratio of concentration of labelled compound and the body fluid compound.
59. The method of claim 58, wherein said derivatization is accomplished by exposing the assayed compound to N-methyl-N(tert-butyl dimethylsylyl) trifluoroacetamide.
60. The method of claim 54, wherein said compound includes deuterated forms of the assayed compound.
61. A method for detecting a deficiency of cobalamin or folic acid in a warm-blooded animal and for distinguishing between a deficiency of cobalamin and a deficiency of folic acid, comprising the steps of:
assaying a first body fluid for an elevated level of cystathionine;
assaying a second body fluid for an elevated level of homocysteine;
correlating said elevated levels of cystathionine and homocysteine with a likelihood of a deficiency of cobalamin or folic acid;
assaying a third body fluid for an elevated level of methylmalonic acid;
assaying a fourth body fluid for an elevated level of 2-methylcitric acid I or 2-methylcitric acid II or both; and
correlating said elevated levels of methylmalonic acid and 2-methylcitric acid I or 2-methylcitric acid II or both with a likelihood of a deficiency of cobalamin but not a likelihood of a deficiency of folic acid.
62. The method of claim 60, wherein said body fluids are serum, urine or cerebral spinal fluid.
63. The method of claim 62, wherein said body fluids are the same.
64. The method of claim 61, wherein said step of assaying the first body fluid for an elevated level of cystathionine includes the steps of:
combining said first body fluid with a compound having cystathionine labelled with an isotope marker; measuring the ratio of concentration of labelled cystathionine and first body fluid cystathionine with a mass spectrometer; and determining the concentration of first body fluid cystathionine present in said first body fluid.
65. The method of claim 64, wherein said first, second, third and fourth body fluids are the same, and a first compound having labelled cystathionine, a second compound having labelled homocysteine, a third compound having labelled methylmalonic acid, and a fourth compound having labelled 2-methylcitric acid I or 2-methylcitric acid II or both, are added to a single sample of said body fluid, and the single sample is divided into a first sample for measuring homocysteine and cystathionine and a second sample for measuring methylmalonic acid and 2-methylcitric acid I or 2-methylcitric acid II or both.
66. The method of claim 64, wherein said step of assaying the fourth body fluid for an elevated level of 2-methylcitric acid I or 2-methylcitric acid II or both includes the steps of:
derivatizing the 2-methylcitric acid I or 2-methylcitric acid II or both before measuring the ratio of concentration of the labelled 2-methylcitric acid I or 2-methylcitric acid II or both and body fluid 2-methylcitric acid I or 2-methylcitric acid II or both.
67. The method of claim 66, wherein said steps of assaying include derivatizing the assayed compound before measuring the ratio of concentration of labelled compound and body fluid compound.
68. The method of claim 67, wherein said derivatization is accomplished by exposing the assayed compound to N-methyl-N(tert-butyl dimethylsylyl) trifluoroacetamide.
69. The method of claim 67, wherein said compounds include deuterated forms of the assayed compound.
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